Loss of Fourth Electron-Transferring Tryptophan in Animal (6–4) Photolyase Impairs DNA Repair Activity in Bacterial Cells

Abstract

(6-4) photolyases [(6-4)PLs] are flavoproteins that use blue light to repair the ultraviolet-induced pyrimidine(6-4)pyrimidone photoproduct in DNA. Their flavin adenine dinucleotide (FAD) cofactor can be reduced to its repair-active FADH form by a photoinduced electron transfer reaction. In animal (6-4)PLs, a chain of four Trp residues was suggested to be involved in a stepwise transfer of an oxidation hole from the flavin to the surface of the protein. Here, we investigated the effect of mutation of the fourth Trp on the DNA photorepair activity of Xenopus laevis (6-4)PL (Xl64) in bacterial cells. The photoreduction and photorepair properties of this mutant protein were independently characterized in vitro. Our results demonstrate that the mutation of the fourth Trp in Xl64 drastically impairs the DNA repair activity in cells and that this effect is due to the inhibition of the photoreduction process. We thereby show that the photoreductive formation of FADH through the Trp tetrad is essential for the biological function of the animal (6-4)PL. The role of the Trp cascade, and of the fourth Trp in particular, is discussed.

Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii

“…Interestingly, in the green lineage including most higher plants and also some algae like O. tauri , the terminal aromatic residue is replaced by a non-redox active phenylalanine (44/542) indicating that the presence of a catalytic tetrad may have gone lost during further evolution and is dispensible at least for plantal (6-4) photolyase activity. Recent studies on the Xl (6-4) photolyase showed that the loss of the fourth redox-active aromatic residue slows indeed photoreduction by up to three orders of magnitude under steady-state conditions and hence impedes recovery of Xl (6-4) photolyase from accidental electron loss of its FAD cofactor (14). …”

“…This intramolecular electron transfer (ET) pathway is elongated in Cr aCRY and related type I animal cryptochromes by a fourth aromatic residue (11,13), which can be either a tyrosine ( Cr aCRY: Y373) or a tryptophan, W394 in the cryptochrome of Drosophila melanogaster . This elongated ET pathway is crucial not only for the photoreduction reactions of aCRY orthologs but also for the function of (6-4) photolyases (14). At least in Cr aCRY, the life time of the terminal Y373-O° radical is unusually long with 26 ms after photoreduction to the FADH° state (13) and even 2.6 s upon light-driven formation of the FADH ― state (11).…”

Sub-nanosecond tryptophan radical deprotonation mediated by a protein-bound water cluster in class II DNA photolyases

“…It is a matter of controversy 35 , 36 whether photoactivation of DNA photolyases through their respective Trp chains is a vital process in vivo or whether the FAD cofactor is naturally and always fully reduced in the living cell, but given that at least two such ET chains have evolved independently in the photolyase-cryptochrome superfamily and survived billions of years of evolution, we dare speculate that the efficient photoactivation of photolyases through the Trp (or Trp/Tyr) chains could be important, especially under intense solar irradiation (which causes simultaneously DNA damage and oxidative stress that could potentially deactivate DNA repair by photolyases by oxidation of their FADH – ). In any case, the electron transfer cascade of three tryptophans and one tyrosine (Trp 381 –Trp 360 –Trp 388 –Tyr 345 in Mm CPDII) is almost strictly conserved within all class II photolyases (Fig.…”

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome

“…The distal tryptophan is usually surface-exposed and can hence be reduced by external electron donors such as thiol compounds 26 or NAD(P)H. 27,28 For animal-like cryptochromes and photolyases, a fourth aromatic residue, Y 373 in CraCRY and W 360 in the photolyase from Xenopus laevis, has been found to act as final electron donor of an extended electron transfer chain. 24,[29][30][31][32] Transient absorption spectroscopy (TAS) has been extensively used to study the mechanistic details of the photoreduction of photolyases 31,[33][34][35][36][37][38][39][40] and cryptochromes, taking advantage of the rather distinct absorption spectra of the species involved ( Figure 1B). 35,39,41 Ultrafast TAS studies have in particular shown that when the flavin is initially oxidized (FAD ox ), the primary electron transfer (ET) takes place in less than one picosecond, 31,35,37,39,41 producing a radical pair of reduced flavin (FAD •-) and oxidized tryptophan (WH •+ ) † .…”